In Human Papillomavirus (HPV) cancers, viral replication pathways involving the HPV oncogenes E6 and E7 are co-opted to promote cancer cell proliferation. However, the altered cellular pathways downstream of these HPV oncogenes remain unclear. Preliminary data indicate that HPV cancers downregulated microRNA (miR)- 203, a miRNA that enforces cell cycle arrest and differentiation and that is also downregulated during HPV replication. Furthermore, HPV oncogenes induced the expression of several transcriptional regulators including EZH2, a methyltransferase known to downregulate the expression of miRNAs. Consistent with a long-term goal of studying pathways causing head and neck cancers (HNSCCs), the objective in this application is to define viral replication pathways downstream of E6 and E7 that regulate the proliferation of HPV-positive cancer cells and that accelerate oral tumor growth. The central hypothesis is that EZH2, which is upregulated by E7, causes the downregulation of miR-203 leading to increased proliferation in partially differentiated tumor cells. Targeting this EZH2-miR-203 pathway will inhibit primary tumor growth and sensitize tumors to radiation. The rationale for this proposed research is to identify pathways involved in viral replication that can be targeted alone or in combination with conventional therapies to more precisely treat HPV-positive cancers. This proposal will develop innovative dual inducible genetically engineered mouse models that sequentially express miR-203 in progressively growing HPV-positive primary oral tumors. In addition, this proposal will use small animal image-guided radiotherapy that models the conventional therapy for HNSCCs. Preclinical observations will be validated using a unique annotated set of human HNSCCs. This proposal is significant because it defines the role of viral replication pathways involving EZH2 and miR-203 that may be targeted to inhibit and to radiosensitize progressively growing oral tumors. This proposal will test the central hypothesis by pursuing three specific aims: (1) determine the HPV oncogenes and downstream transcriptional regulators that inhibit miR-203 expression; (2) identify cellular mechanisms by which restoring miR-203 expression inhibits progressive oral tumor growth and increases sensitivity to DNA damage; and (3) identify cellular mechanisms by which inhibiting EZH2 alters progressive oral tumor growth and response to DNA damage. Aim 1 will use targeted siRNA screens and RNA sequencing to identify regulators of miR-203. Aim 2 will restore miR-203 expression in progressively growing oral tumors in order to determine the extent to which a single miRNA is sufficient to inhibit tumor cell proliferation and to cause radiosensitivity. Aim 3 will delete EZH2 in primary HPV- tumors in order to determine the necessity of this methyltransferase on miR-203 expression and tumor cell proliferation. Ultimately, these results will facilitate new therapeutic strategies targeting viral replication pathways in order to treat HPV-positive HNSCCs and, possibly, in HPV-negative HNSCCs and other HPV- associated cancers, in general.